We propose to develop a novel and innovative system for studying very early stages of colon cancer development. This in vitro culture system involves promoting embryonic stem cell (ESC) differentiation into intestinal organoids through a definitive endoderm intermediate, and then initiating cell transformation through oncogene expression or tumor suppressor gene knockdown. This experimental system, which we have been developing with mouse and human ESCs, is anticipated to be robust and highly flexible and enable cancer biologists to address fundamental questions relating to very early events in colon carcinogenesis. Advantages of this system include an in vitro format compatible with the rapid analysis of nutrients, small molecules, siRNAs or other biologics. Furthermore, intestinal organoids can be generated from human ESCs, allowing the detailed study of intestinal cells from individuals with distinct colon cancer risks. In the proposed studies we will assess the utiliy of intestinal organoids for studying the effects of Braf oncogene activation. Our interest in Braf mutated cells stems from the fact that BRAF-mutated cancers frequently develop in the human proximal colon where they can be difficult to detect and remove, making effective chemoprevention of these cancers an important goal. Intestinal organoids will be prepared from mouse ESCs in which Braf oncogene become activated during intestinal lineage commitment (using Villin- Cre/LSL-BrafV600E mice) and are regulated by their native promoters. We will then evaluate how oncogene expression influences the organoids in relationship to endogenous intestinal tissues in vivo. This work will focus on oncogene effects on cellular turnover dynamics, intestinal stem cell expansion and cellular differentiation. Since Braf activation is associated wih epigenetic alterations, we will also determine the effect of oncogene activation on the expression of epigenetic modifying factors and the silencing of growth regulatory genes. We will also determine the ability of cell growth modulating chemopreventive agents to correct molecular and proliferation defects in oncogene-expressing intestinal organoids. Finally, we will develop high throughput assay systems to screen natural compound and pharmacological agent libraries for novel chemopreventive agents that selectively inhibit the growth of Braf-expressing intestinal cells. We anticipate indentifying novel chemopreventive agents (or agent combinations) for further development in subsequent proposals. Finally, establishing the organoid system for cancer research could facilitate the study many factors and variables that impact cancer promotion and prevention. PUBLIC HEALTH RELEVANCE: The KRAS and BRAF oncogenes can be activated early in colon carcinogenesis and stimulate a complex combination of pro-growth and anti-growth signaling pathways that ultimately determine whether the cells can progress to colon cancer or not. In this proposal we will derive intestinal cells from embryonic stem cells to establish a simple system to study the complex events that follow KRAS and BRAF oncogene activation. Our focus will be to determine the role of epigenetics in promoting the growth effects of the oncogenes with an eye towards understanding how the pro-growth signaling might be suppressed for colon cancer prevention.